Computational protocol: Tubulin tyrosine nitration regulates microtubule organization in plant cells

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Protocol publication

[…] Three-dimensional (3D) model of plant kinesin-8 was constructed on the online server SwissModel Workspace (Arnold et al., ) using the A. thaliana primary sequence (UniProtKB/TrEMBL code: Q9FZ77) and 3LRE crystal of human kinesin-8 motor domain KIF18A (Peters et al., ) as a matrix. The full 3D models of α-and β-tubulins from A. thaliana were constructed using the online server I-TASSER on the basis of primary sequences P11139 and P12411, respectively (Blume et al., ). Overlay of the constructed molecules of A. thaliana kinesin-8, α- and β-tubulins with the appropriated structures from 1IA0 crystal (Kikkawa et al., ) was performed in Swiss-PdbViewer 4.1.0 software package. Alteration of C-terminal region position of plant α-tubulin and Tyr450 modifications were simulated in the Discovery Studio 3.5 Visualizer company Accelrys© software package.The optimization of the molecular geometry of the constructed models, molecular dynamics simulation of protein-protein complex and analysis of the results were performed using the specialized modules from Gromacs 4.5.4 software package (Hess et al., ; Pronk et al., ). Topology data for the unnatural amino acid used for charmm27 force field was obtained from the database SwissSidechain (Gfeller et al., ). In order to reproduce the intracellular environment, protein complexes were immersed in water and its volume was determined by the size of the studied macromolecules. Distant electrostatic interactions were calculated by PME method (Particle Mesh Ewald) (Essmann et al., ). As one of the conditions of its use for the calculation of the Coulomb interactions is the neutral system status, the nonzero charges of the obtained systems were neutralized by the automatic substitution of random water molecules with sodium and chlorine ions, which amount corresponded to physiological concentration (0.15 mol/L). Optimization of the molecular geometry was carried out by the potential energy minimization by the steep descent algorithm under the maximum steps number of 1000 and gradient 0.1 in the force field charmm27. The quality of the obtained models was estimated on the MolProbity server (Chen et al., ).The process of the molecular dynamics that lasted 20 ns was carried out using grompp and mdrun modules. To simulate the solvent volume periodic boundary conditions were applied. The temperature of the system was maintained at 310K using the Berendsen thermostat with the interaction time of 0.1 ps. Constant pressure was maintained by an external barostat. Bond lengths between the hydrogen atoms were fixed at the equilibrium levels using the Lincs algorithm (Hess et al., ). Error messages in the molecules structure were absent. Analysis of the molecular dynamics was carried out using g_dist and g_hbond modules. Visualization of all molecules was performed in the Discovery Studio 3.5 Visualizer company Accelrys© software package.Molecular dynamics calculation was carried out with the assistance of The Ukrainian National Grid (UNG) (http://grid.nas.gov.ua) and IFBG cluster of virtual organization CSLabGrid. […]

Pipeline specifications

Software tools I-TASSER, Swiss-PdbViewer, GROMACS, MolProbity, P-LINCS
Databases UniProtKB
Organisms Arabidopsis thaliana, Nicotiana tabacum
Chemicals Nitric Oxide